1. Field of the Invention
This invention is related in general to the field of microscopy. In particular, it relates to a method and apparatus for finding the best-focus position of a scanning array microscope that includes a plurality of optical imaging elements with respective optical axes.
2. Description of the Related Art
The object to be imaged by a microscope is typically located at the object plane by being placed on a substrate that is, in turn, positioned on a stage of the microscope that can be moved laterally with respect to the optical axis of the lens system. The stage may be motorized so that this movement may be automated or controlled by a computer. Moreover, the image plane may be provided with a camera or other imaging device for recording the image, or for monitoring the image under the same computer control.
In addition to being characterized by its numerical aperture, an imaging lens system is also characterized by its field of view. The field of view in visible light microscopes typically ranges from tens of microns to a few millimeters. This means that a macroscopically sized object of 20 mm×50 mm, for example, requires many movements of the stage for imaging the entire object. The stage manipulation and the consequent time required to image an object under high magnification is particularly troublesome in pathology analysis because the diagnostic information in the tissue may be located in only a small portion of the object that is being imaged.
A recent innovation in the field of light microscopy addresses this problem using a miniaturized microscope array which, when applied to a common object, is also referred to as an “array microscope.” As described in commonly owned PCT/US02/08286, herein incorporated by reference, each miniaturized microscope includes a plurality of optical elements individually positioned with respect to a corresponding image plane and configured to image respective sections of the object. The array further includes a plurality of image sensors corresponding to respective optical elements and configured to capture image signals from respective portions of the object.
In such an array microscope, a linear array of miniaturized microscopes is preferably provided with adjacent fields of view that span across a first dimension of the object (also referred to herein as y direction), and the object is translated past the fields of view across a second dimension (x direction) to image the entire object. Because each miniaturized microscope is larger than its field of view (having respective diameters of about 1.8 mm and 200 μm, for example), the individual microscopes of the imaging array are staggered in the direction of scanning so that their relatively smaller fields of view are offset over the second dimension but aligned over the first dimension, as illustrated in FIG. 1. Thus, the detector array provides an effectively continuous linear coverage along the first dimension which eliminates the need for mechanical translation of the microscope in that direction, providing a highly advantageous increase in imaging speed by permitting complete coverage of the sample surface with a single scanning pass along the second dimension.
As always in microscopy, the value of a procedure is a function of the quality of the images produced by it. Thus, the ability to maintain a sharp image during scanning of the test object through the microscope is essential for obtaining good-quality results. This is particularly difficult to achieve when high numerical-aperture microscopic imaging is used, which is characterized by small depths of field often in the order of less than 1 μm. Moreover, in typical biological microscopy, such as used in the field of pathology, the sample material is deposited on a transparent slide and covered with a layer of fixing medium and a cover glass. Often these samples exhibit significant variations in the thickness of the tissue, the fixing medium, and the cover glass, which requires different axial positions of the microscope for best-focus imaging of different parts of the sample. For example, the deviation from perfect flatness of the slides used in biological microscopy is by itself often greater than the depth of focus of the imaging optics. Therefore, a scan of the entire slide at a fixed focal distance necessarily produces variations in the image quality between different sections of the slide, which may require refocusing during a scan in function of the location of the image of interest on the slide.
In order to avoid refocusing of the imaging optics during a scan, a pre-scan run is often performed with conventional microscopes in order to acquire data used to find best-focus locations for different sections of the test slide. These locations are then used to guide the subsequent scanning process for that particular slide. In general, these prior-art pre-scan systems are based on the concept of selecting a number of points on the test surface, either randomly or according to judiciously selected criteria, and finding the best-focus position for each such point. As is well understood in the art, best focus may be determined by a variety of methods, such as, for instance, by finding the optical-axis position that produces maximum contrast or by analyzing the high-frequency content of the signal at each point. The information is then used in some manner to construct a best trajectory for the scanning operation. For instance, U.S. Publication No. 2002/0090127 teaches the development of a focus surface based on the best-focus position of at least three points on the test surface. Inasmuch as the implementation of this procedure with conventional microscopes requires positioning of the optical system in front of each selected point and searching for the best-focus axial position at each location, this approach is slow and not well suited for parallel-imaging devices such as the array microscopes described in PCT/US02/08286.
In view of the unsuitability and shortcomings of the prior-art focusing methods when applied to array microscopes, it would be very desirable to provide an approach that affords the flexibility of operating within the entire range of the sample surface without loss of continuity, precision or resolution. This invention provides a novel solution to that end.